© 2002 Oxford University Press
Nucleic Acids Research, 2002, Vol. 30, No. 7 e32
Glass bead purification of plasmid template DNA for high throughput sequencing of mammalian genomes Debra A. Dederich, Geoffrey Okwuonu, Toni Garner, Amanda Denn, Angelica Sutton, Michael Escotto, Ashley Martindale, Oliver Delgado, Donna M. Muzny, Richard A. Gibbs and Michael L. Metzker* Human Genome Sequencing Center, Department of Molecular and Human Genetics, Baylor College of Medicine, 1 Baylor Plaza, Alkek Building, N1409, Houston, TX 77030, USA Received October 10, 2001; Revised and Accepted February 9, 2002
ABSTRACT To meet the new challenge of generating the draft sequences of mammalian genomes, we describe the development of a novel high throughput 96-well method for the purification of plasmid DNA template using size-fractionated, acid-washed glass beads. Unlike most previously described approaches, the current method has been designed and optimized to facilitate the direct binding of alcohol-precipitated plasmid DNA to glass beads from alkaline lysed bacterial cells containing the insoluble cellular aggregate material. Eliminating the tedious step of separating the cleared lysate significantly simplifies the method and improves throughput and reliability. During a 4 month period of 96-capillary DNA sequencing of the Rattus norvegicus genome at the Baylor College of Medicine Human Genome Sequencing Center, the average success rate and read length derived from >1 800 000 plasmid DNA templates prepared by the direct lysis/glass bead method were 82.2% and 516 bases, respectively. The cost of this direct lysis/glass bead method in September 2001 was ∼10 cents per clone, which is a significant cost saving in high throughput genomic sequencing efforts. INTRODUCTION Rapid and efficient methods for nucleic acid purifications are essential for many molecular biology applications. Several research groups have described methods for the purification of plasmid DNA that produce templates of sufficient quality for DNA sequence applications (1–12). The introduction of capillary electrophoresis DNA sequencers, however, has put further demands on template purity when compared to automated slab gel systems. For high throughput DNA sequencing of whole mammalian genomes, purification methods must also be designed to meet the challenges of platform flexibility, increasing scalability and decreasing costs, while maintaining high quality. At the Baylor College of Medicine Human
Genome Sequencing Center (BCM-HGSC), the effort required to generate a draft sequence of the Rattus norvegicus genome in a 2 year period (13) required a departure from the M13 DNA sequencing approach and development of a higher throughput, paired end plasmid DNA sequence strategy. The majority of plasmid preparation protocols rely on the ‘alkaline lysis’ method, which uses a narrow pH range (12.0–12.5) to selectively denature linear, but not covalently closed circular DNA (1). The alkaline lysate is rapidly neutralized to form an insoluble aggregate consisting of genomic DNA, protein–detergent complexes and high molecular weight RNA. In the original method the cleared lysate is then carefully recovered by centrifugation and ethanol precipitated for isolation of crude plasmid DNA. Alternative methods, such as the boiling method (2) and microwave preparation (11), have been used to isolate plasmid DNA; however, the alkaline lysis method represents the most robust and stable platform for further development of high throughput purification methods. To improve the purity of plasmid DNA, several innovations have been coupled to the step of processing the cleared lysate. These methods include agarose gel electrophoresis extraction, column chromatography, cesium chloride gradient centrifugation, and selective adsorption using solid phase supports. Of these strategies, the latter has the advantages of simplicity and parallel processing via multiplexing of samples. The required centrifugation step in preparing the cleared lysates, however, significantly hinders automation by robotics. A filtration or clearing plate (9) or selective binding conditions to carboxylated magnetic particles (12) have been used to remove the insoluble aggregate material from the soluble plasmid DNA; however, these clearing steps have the disadvantage of increasing costs in materials and efforts, which decrease the cycle time of the method. Glass particles, powder and beads have proven useful for purifying nucleic acids. An early technique to isolate DNA from agarose gels involved the use of the chaotropic salts sodium iodide (14) and sodium perchlorate (15,16) to facilitate binding of the DNA to common silicate glass, flint glass and borosilicate glass (glass fiber filter). Plasmid DNA initially isolated as a cleared lysate has also been purified by binding to glass fiber filter powder in the presence of sodium perchlorate (3). Recently, Engelstein et al. described the purification of plasmid DNA under high sodium chloride and 10% polyethylene
*To whom correspondence should be addressed. Tel: +1 713 798 7565; Fax: +1 713 798 5741; Email:
[email protected]
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glycol (PEG) conditions with silica particles (9). All of these methods have the common element of high salt solutions, in which the adsorption of plasmid DNA onto the glass substrate occurs most likely by a mechanism similar to adsorption chromatography. In the method presented in this article we utilize a direct lysis protocol, in which the lysate containing the insoluble aggregate is directly added to a 96-well filter plate containing binding solution and glass beads. Unlike the high salt methods, we have found that alcohol precipitates of plasmid DNA bind efficiently to glass substrates and can occur in the presence of the insoluble cellular aggregate lysate. The methods developed here, which allow compatibility of the direct lysis solution with plasmid DNA binding to glass beads, lends itself to a simplified high throughput method and has the flexibility for complete automation on standard robotic platforms. The cost of each plasmid DNA prepared by the glass bead method is currently ∼10 cents, which affords significant advantages in high throughput sequencing of mammalian genomes. Considering genome center discounts, this represents a 4-fold saving in reagents and materials and an ∼10-fold saving at market prices. MATERIALS AND METHODS Reagents and supplies The reagents necessary to perform the glass bead preparation method are readily available as standard components, such as glucose, Tris, EDTA, NaOH, IGEPAL CA-630 (formerly known as Nonidet P-40), potassium acetate, glacial acetic acid and 2-propanol (EM Science, Gibbstown, NJ) or as pre-made solutions I, II and III (catalog nos VW8869-1, VW8870-1 and VW8871-1, respectively; EM Science). Pure (100%) ethanol was purchased from Aaper (Shelbyville, KY). Solutions I–III are composed of, respectively: 51 mM glucose, 26 mM Tris–HCl, pH 8.0, and 51 mM EDTA; 0.9 N NaOH and 0.125% IGEPAL CA-630; and 387 mM potassium acetate, 15% glacial acetic acid. RNase A (Life Technologies, Rockville, MD) was added fresh to solution I (final concentration 0.35 mg/ml) prior to the lysis step. Glass beads were purchased as 212–150 µm sizefractionated, acid-washed glass beads from Sigma (St Louis, MO) or EM Science or as 1 800 000 plasmid DNA templates have been prepared using this direct lysis/glass bead preparation method (Table 1). The average success rate over this 4 month period was 82.2%, which was calculated as the percentage of sequencing reactions yielding a
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Figure 2. Snapshot images of ABI model 3700 capillary array views from two different chromatographic regions from a rat BAC clone skimmed project. The vertical chromatogram to the left is partially processed fluorescence data from capillary number 71 showing good baseline resolution of fluorescent bands near 600 bases. The signal reduction in the later region is the result of electrokinetic injection of sequencing reactions into capillaries.
minimum of 100 Phred bases with quality values ≥20 (Q20). The average read lengths for successful sequencing reactions
were calculated by counting the total number of Q20 bases and dividing by the number of sequencing reads. The average read
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Table 1. Summary statistics of the BCM-HGSC rat genome sequence effort using the glass bead preparation between 12 May and 15 September 2001 Month (2001)
No. of sequencing reactions loaded
Pass quality (%)
Read length (Q20 bases)
May
738 256
80.2
503
Junea
455 441
79.8
491
July
684 342
82.4
506
1 306 970
83.5
534
444 236
84.2
529
3 619 245
82.2
516
August September Total
Reactions were loaded on 69 Applied Biosystems Model 3700 DNA sequencers, which included both WGS and BAC clone shotgun skim sequence reactions. The number of plasmid DNA templates required to generate the total number of paired end sequence reactions is half (∼1 810 000). DNA sequencing quality was determined using the base caller Phred (18) and the average success rates are reported as a percentage of sequencing reactions, which produced a minimum of 100 Q20 bases, and the average read lengths of successful reads are derived from the total number of Q20 bases divided by the number of sequencing reads. aLower production numbers were the result of tropical storm Allison hitting Houston, Texas on 8 June 2001.
length for the entire data set was 516 bases, with the latter 2 months yielding even better success rates and read lengths. Overall, these data show good quality and reproducibility of plasmid DNA templates prepared using the direct lysis method for high throughput capillary DNA sequencing of the rat genome. The development of a simplified and robust method for plasmid DNA templates described here meets the challenges for high throughput sequencing of whole mammalian genomes. Automation and increased well number per plasmid plate can achieve additional increases in throughput. Elimination of the cleared lysate step simplifies the method and streamlines the process from pelleted bacterial cells in growth boxes to highly purified plasmid DNA templates in a completely automated manner without interruption. Currently we are evaluating the performance of the direct lysis/glass bead preparation method on a commercial robotic platform at the BCM-HGSC. Moreover, our sequencing pipeline is currently configured to combine two 96-well template plates into one 384-well sequencing plate for paired end sequencing of plasmid DNA, which is potentially problematical in that it can mix different BAC clone projects. While a 384-well format is useful for DNA sequencing, we are investigating the feasibility of a 192-well template format. Besides the advantage of doubling throughput, the 192-well format has the additional benefit of simplifying the tracking of multiple BAC clone projects by single plate-to-plate transfers for paired end sequencing.
ACKNOWLEDGEMENTS We are grateful to a number of BCM-HGSC staff members, including George Weinstock, Erica Sodergren, Graham Scott, Paul Havlak, Lora Lewis, Stephanie Moore, Shereen Thomas, Carrie Burkett and Hailey Stanley, for their technical expertise in their respective roles in the BCM-HGSC DNA production– sequencing pipeline. This work was supported by grants 1-U54-HG02139 and 1-U54-HG02345 from the National Institutes of Health. REFERENCES 1. Birnboim,H.C. and Doly,J. (1979) A rapid alkaline extraction procedure for screening plasmid DNA. Nucleic Acids Res., 7, 1513–1523. 2. Holmes,D.S. and Quigley,M. (1981) A rapid boiling method for the preparation of bacterial plasmid. Anal. Biochem., 114, 193–197. 3. Marko,M.A., Chipperfield,R. and Birnboim,H.C. (1982) A procedure for the large-scale isolation of highly purified plasmid DNA using alkaline extraction and binding to glass powder. Anal. Biochem., 121, 382–387. 4. Zhou,C., Yang,Y. and Jong,A.Y. (1990) Mini-prep in ten minutes. Biotechniques, 8, 172–173. 5. Huang,G.M., Wang,K., Kuo,C., Paeper,B. and Hood,L. (1994) A high-throughput plasmid DNA preparation method. Anal. Biochem., 223, 35–38. 6. Hawkins,T.L., O’Connor-Morin,T., Roy,A. and Santillan,C. (1994) DNA purification and isolation using a solid-phase. Nucleic Acids Res., 22, 4543–4544. 7. Ng,W., Schummer,M., Cirisano,F.D., Baldwin,R.L., Karlan,B.Y. and Hood,L. (1996) High throughput plasmid mini preparations facilitated by micro-mixing. Nucleic Acids Res., 24, 5045–5047. 8. Itoh,M., Carninci,P., Nagaoka,S., Sasaki,N., Okazaki,Y., Ohsumi,T., Muramatsu,M. and Hayashizaki,Y. (1997) Simple and rapid preparation of plasmid template by a filtration method using microtiter filter plates. Nucleic Acids Res., 25, 1315–1316. 9. Engelstein,M., Aldredge,T.J., Madan,D., Smith,J.H., Mao,J.-I., Smith,D.S. and Rice,P.W. (1998) An efficient, automatable template preparation for high throughput sequencing. Microbial Comp. Genomics, 3, 237–241. 10. Itoh,M., Kitsunai,T., Akiyama,J., Shibata,K., Izawa,M., Kawai,J., Tomaru,Y., Carninci,P., Shibata,Y., Ozawa,Y., Muramatsu,M., Okazaki,Y. and Hayashizaki,Y. (1999) Automated filtration-based high-throughput plasmid preparation system. Genome Res., 9, 463–470. 11. Marra,M.A., Kucaba,T.A., Hillier,L.W and Waterston,R.H. (1999) High-throughput plasmid DNA purification for 3 cents per sample. Nucleic Acids Res., 27, e37. 12. Elkin,C.J., Richardson,P.M., Fourcade,H.M., Hammon,N.M., Pollard,M.J., Predki,P.F. Glavina,T. and Hawkins,T.L. (2001) High-throughput plasmid purification for capillary sequencing. Genome Res., 11, 1269–1274. 13. Marshall,E. (2001) Rat genome spurs an unusual partnership. Science, 291, 1872. 14. Vogelstein,B. and Gillespie,D. (1979) Preparative and analytical purification of DNA from agarose. Proc. Natl Acad. Sci. USA, 76, 615–619. 15. Yang,R., Lis,J. and Wu,R. (1979) Elution of DNA from agarose gels after electrophoresis. Methods Enzymol., 68, 176–182. 16. Chen,C.W. and Thomas,C.A.,Jr (1980) Recovery of DNA segments from agarose gels. Anal. Biochem., 101, 339–341. 17. Andersson,B., Wentland,M.A., Ricafrente,J.Y., Liu,W. and Gibbs,R.A. (1996) A “double adaptor” method for improved shotgun library construction. Anal. Biochem., 236, 107–113. 18. Ewing,B., Hillier,L., Wendl,M.C. and Green,P. (1998) Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res., 8, 175–185.
